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1 Comprehensive Cancer Center and Cancer Research Institute, University of California at San Francisco, San Francisco, California, United States of
America, 2 Canary Foundation, San Jose, California, United States of America, 3 Department of Microbiology and Immunology, University of California
at San Francisco, San Francisco, California, United States of America, 4 Department of Pediatrics, University of California at San Francisco, San
Francisco, California, United States of America
Background. Among gynecologic cancers, ovarian cancer is the second most common and has the highest death rate. Cancer
is a genetic disorder and arises due to the accumulation of somatic mutations in critical genes. An understanding of the
genetic basis of ovarian cancer has implications both for early detection and for therapeutic intervention in this population of
patients. Methodology/Principal Findings. Fifteen ovarian cancer cell lines, commonly used for in vitro experiments, were
screened for mutations using bidirectional direct sequencing in all coding regions of BRAF, MEK1 and MEK2. BRAF mutations
were identified in four of the fifteen ovarian cancer cell lines studied. Together, these four cell lines contained four different
BRAF mutations, two of which were novel. ES-2 had the common B-Raf p.V600E mutation in exon 15 and Hey contained an
exon 11 missense mutation, p.G464E. The two novel B-Raf mutants identified were a 5 amino acid heterozygous deletion
p.N486-P490del in OV90, and an exon 4 missense substitution p.Q201H in OVCAR 10. One of the cell lines, ES-2, contained a
mutation in MEK1, specifically, a novel heterozygous missense substitution, p.D67N which resulted from a nt 199 GRA
transition. None of the cell lines contained coding region mutations in MEK2. Functional characterization of the MEK1 mutant
p.D67N by transient transfection with subsequent Western blot analysis demonstrated increased ERK phosphorylation as
compared to controls. Conclusions/Significance. In this study, we report novel BRAF mutations in exon 4 and exon 12 and
also report the first mutation in MEK1 associated with human cancer. Functional data indicate the MEK1 mutation may confer
alteration of activation through the MAPK pathway. The significance of these findings is that BRAF and MEK1/2 mutations may
be more common than anticipated in ovarian cancer which could have important implications for treatment of patients with
this disease and suggests potential new therapeutic avenues.
Citation: Estep AL, Palmer C, McCormick F, Rauen KA (2007) Mutation Analysis of BRAF, MEK1 and MEK2 in 15 Ovarian Cancer Cell Lines: Implications
for Therapy. PLoS ONE 2(12): e1279. doi:10.1371/journal.pone.0001279
RESULTS
BRAF and MEK1 Mutations
Genomic DNA from 15 ovarian cancer cell lines was screened for
BRAF mutations in coding exons 1–18. Four mutations were
identified in four individual cell lines: OVCAR 10, OV90, Hey
and ES-2 (Figure 1). None of the other cell lines had BRAF
mutations. Two of the four BRAF mutations identified were novel.
OVCAR 10 contained a nt 603 GRT transversion causing a
heterozygous missense substitution p.Q201H in exon 4 (Figure 1A).
OV90 contained a novel heterozygous 5 amino acid deletion,
p.N486-P490del, in exon 12 (Figure 1B). In addition to the two
novel BRAF mutations identified, two additional mutations which
have been previously reported in cancer were identified. Hey
contained a nt 1391 GRA transition resulting in an exon 11
missense mutation, p.G464E (Figure 1C). The electropherogram
demonstrated loss of heterozygosity at this locus. ES-2 contained
an exon 15, TRA transversion at nt 1799, substituting glutamic
acid for valine at position 600 (p.V600E) (Figure 1D). None of
these mutations were identified in the controls.
All eleven coding exons of MEK1 and MEK2 were also sequenced
in the same cell lines and controls. One mutation in MEK1 was
identified in ES-2 consisting of a novel heterozygous missense
substitution, p.D67N, which resulted from a nt 199 GRA transition
(Figure 1E). No other nonsynonymous substitutions in MEK1 were
identified. All eleven coding exons of MEK2 were sequenced and no
nonsynonymous substitutions were identified.
Nucleotide Variation
In addition to these mutations, a total of four different synonymous
single nucleotide polymorphisms (SNPs) were identified in BRAF
(Table 1), MEK1 (Table 2), and MEK2 (Table 3). Three of these
four SNPs were found in five or more of the fifteen cell lines and
have been previously reported (www.ncbi.nlm.nih.gov/SNP/). In
order of frequency, the three synonymous database SNPs include:
i) MEK2 p.I220I (rs10250) present in 11 of the 15 cell lines (73%),
ii) BRAF p.G634G (rs9648696) found in six of the 15 cell lines
(40%) and, iii) MEK2 p.D151D (rs17851657) identified in five of
the 15 cell lines (33%). There was one uniquely identified MEK1
SNP in OVCAR 10 resulting from a nt 348 heterozygous GRA
transition in exon 3, p.Q113Q (Table 2). All synonymous SNPs
were characterized by SIFT (Sorting Intolerant From Tolerant)
and determined to be tolerated.
...........................................................
Table 1. BRAF sequence variations identified in ovarian cancer cell lines.
..................................................................................................................................................
Cell Line Exon Nucleotide Mutation Amino Acid Mutation dbSNP* Predicted Protein Function**
*
www.ncbi.nlm.nih.gov/SNP/
**
blocks.fhcrc.org/sift/SIFT.html
doi:10.1371/journal.pone.0001279.t001
...................................
*
www.ncbi.nlm.nih.gov/SNP/
**
blocks.fhcrc.org/sift/SIFT.html
doi:10.1371/journal.pone.0001279.t003
functionally affected. To corroborate this information, MEK1 characterization of the sensitivity of various BRAF and MEK
p.D67N was transiently transfected into HEK 293T cells, and mutants to small molecule inhibition is an important avenue to
ERK phosphorylation was measured by Western blot analysis. pursue towards the development of effective treatments for ovarian
The MEK1 p.D67N mutant is activating as demonstrated by cancer.
increased ERK phosphorylation as compared to empty vector and
wildtype MEK1, two controls which do not activate ERK. METHODS
However, the level of ERK phosphorylation for p.D67N is less
than the positive control CFC MEK1 p.Y130C mutant [18]. Cell Lines and Isolation of Genomic DNA
In addition to BRAF and MEK1 mutations, several synonymous Fifteen ovarian cancer cell lines, commonly used for in vitro
database SNP were also identified. B-Raf p.G634G (rs9648696) experiments, were screened for mutations: OVCAR 3, SKOV 3,
was identified in 40% of the 15 cell lines, MEK2 p.I220I (rs10250) TOV-112d, A2780, OV90, ES-2, TOV-21g, Caov-3, A1847,
was present in 73% and MEK2 p.D151D (rs17851657) was IGROV 1, OVCAR 5, 2008, OVCAR 10, PEO1 and Hey. Cell
identified in 33% of the cell lines. These synonymous database pellets from each of these cell lines were kindly provided by Drs.
SNPs were present at a frequency which is comparable to that Charles Drescher and Beatrice Knudsen. Genomic DNA was
previously reported (http://www.ncbi.nlm.nih.gov/SNP/snp; isolated from cell pellets using the QIAamp DNA Tissue kit
[43]). There was one uniquely identified MEK1 SNP in OVCAR (Qiagen, Valencia, CA), according to the manufacturer’s instruc-
10 resulting from a nt 348 heterozygous GRA transition in exon tions. Forty healthy human controls were included in this study.
3, p.Q113Q. All synonymous SNPs were characterized by SIFT Genomic DNA was isolated from peripheral blood lymphocytes
and determined to be tolerated. using the QIAamp DNA Blood Midi kit (Qiagen, Valencia, CA),
Our findings emphasize that mutations which alter function of according to the manufacturer’s instructions. Control samples
the MAPK pathway play an important role in ovarian cancer [15]. were obtained under an approved institution review board from
In addition, the identification of mutations in key MAPK pathway the University of California San Francisco.
components will be important in determining the responsiveness of
the cancer to therapeutics, the aggressive and metastatic behavior PCR and Bidirectional Sequencing
of the tumor and the prognosis of the patient. Four of 15 (26%) cell PCR primers were designed to amplify all coding exons and
lines in this study had BRAF mutations and 1 of 15 (7%) had a intronic flanking regions of BRAF (NM_004333.2), MEK1
MEK mutation. It is known that BRAF mutations have been (NM_002755.2) and MEK2 (NM_030662.2) (Table 4 and
identified in certain types of ovarian cancer; however, mutations in Table 5). For sequencing, the PCR primers were modified on
the downstream effectors of B-Raf including MEK1 and MEK2 the 59 end to include M13 forward (GTAAAACGACGGC-
may also be important contributors of ovarian cancer tumorigen- CAGT) and reverse (CAGGAAACAGCTATGACC) sequences.
esis. Defining the pathogenetics of ovarian cancer may enable the PCR and sequencing were performed by Agencourt Bioscience
use of targeted therapeutics, such as small molecule inhibitors of Corporation (Beverly, MA). Bidirectional sequencing was con-
MAPK pathway, which have recently begun to demonstrate great ducted with ABI BigDye v3.1 Cycle Sequencing Kit (Applied
promise [16]. In addition, a report indicates that cells with Biosystems, Foster City, CA) according to manufacturer’s
activated B-Raf have enhanced, selective sensitivity to MEK guidelines and run on an ABI3730xl capillary sequencing
inhibitors [44]. Our results underscore the importance that further instrument (Applied Biosystems, Foster City, CA).
...................................................................................
doi:10.1371/journal.pone.0001279.t004
...............................................................................................
Table 5. MEK1 and MEK2 Sequencing Primers.
..................................................................................................................................................
Gene Exon Forward Reverse bp
doi:10.1371/journal.pone.0001279.t005
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